Disease caused by the obligate intracellular parasite Toxoplasma gondii is inherently linked to fast replication cycles of the tachyzoite life stage. Tachyzoite division produces two daughters per division round, which are assembled within the mother cell through a unique 'internal budding' process. Assembly of the peripheral cytoskeleton drives budding and serves as a scaffold for organelle assemblage and segregation. The cytoskeleton is composed of microtubules together with two unique elements not shared with the host (intermediate filaments in complex with flattened vesicles together called the inner membrane complex or IMC). This proposal aims to characterize a unique component of the replication machinery, the intermediate filament cytoskeleton. To date, four different filament genes have been studied (TgIMC1-4), but a preliminary genomic survey identified 16 genes in total. Preliminary studies of several filaments throughout development identified a surprising spectrum in behavior and hint at a thus far underappreciated sophistication of the intermediate cytoskeleton. Therefore the research team proposes to assess the localization, post-translation modification, and proteolytic processing of all 16 genes throughout parasites division (Aim 1). Furthermore, preliminary data identifies protein TgMORN1 as a central organizer between two filament structures (the IMC and the posterior cup), which also provides a scaffold for the constriction of the cytoskeleton during maturation. The pivotal role of TgMORN1 will be further dissected by identifying the proteins in the large TgMORN1 complex (Aim 2). Finally, we want to construct a model of the process wherein morphological changes during the division process can be linked to biochemical events (Aim 3). Upon completion of this proposal, understanding on the who, when, where and how of the assembly, maturation, stability and turnover of the intermediate filament cytoskeleton will allow predictions on how to best interfere with division, providing a rational basis for development of new therapeutics to treat toxoplasmosis. PUBLIC HEALTH RELEVANCE: Limited treatment options are available to treat disease caused by Apicomplexa. We propose to study the internal budding process of the model parasite Toxoplasma gondii, in particular the contribution of the intermediate filament cytoskeleton, and identify the (unique) proteins in the cell division machinery providing potential new specific drug targets.